Liquid Chromatography/Mass Spectrometry of Carotenoids Using Atmospheric Pressure Chemical Ionization

Abstract

Carotenoids contained in plant extracts were analyzed using liquid chromatography/positive-and negative-ion atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) with a narrow-bore C30 reversed-phase high-performance liquid chromatographic (HPLC) column and a gradient solvent system containing methanol–methyl tert-butyl ether–ammonium acetate at a flow rate of 300 μl min-1. In addition to mass spectro-metric detection, photodiode-array UV/visible absorbance detection was used between the HPLC column and mass spectrometer for additional carotenoid characterization. Positive-ion APCI produced protonated molecules and molecular ions for both xanthophylls and, unexpectedly, hydrocarbon carotenes; and during negative-ion APCI, M-• and [M-H]- ions were observed. In order to investigate the origin of the unexpected [M+H]+ ions, positive-ion APCI of β-carotene was investigated using deuterochloroform as the only solvent. Because β-carotene formed primarily deuterated ions, [M+D]+, during APCI in deuterochloroform, the mobile phase was determined to be the source of hydrogen for protonation. The hydroxylated xanthophyll lutein fragmented during positive-ion APCI to eliminate water from the protonated molecule and form the base peak of m/z551. Using collision-induced dissociation in the ion source, additional fragmentation pathways characteristic of tandem mass spectra of carotenoids were observed such as retro-Diels–Alder fragmentation, [M-56]+•, for α-carotene and loss of toluene from the molecular ion, [M-92]+• for lutein, α-and β-carotene. The limits of detection for protonated molecules of α-carotene and lutein were approximately 3 and 13 pmol, respectively. In negative-ion APCI, the limits of detection were approximately 3 and 1 pmol for M-• ions of α-carotene and lutein, respectively.